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At least 1,225 records · Page 68Linked to original sources
PubMed1992

Staining of argininosuccinate lyase activity in polyacrylamide gel.

A procedure for the direct staining of argininosuccinate lyase activity in polyacrylamide gel is described. The method was based on coupling one of the enzymatic products fumarate with fumarase and malic enzyme catalyzed reactions. Fumarate was first converted to L-malate by fumarase. Malic enzyme then catalyzed the oxidative decarboxylation of L-malate to give CO2 and pyruvate with concomitant reduction of NADP+ to NADPH. Finally the reducing power of NADPH was coupled to phenazine methosulfate and in turn to nitroblue tetrazolium yielding a deeply colored insoluble formazan which may be quantitized or semiquantitized by densitometer.

Animals↗
PubMed1983

Solubilization and separation of divalent cation dependent adenosinetriphosphatase in native gradient polyacrylamide slab gel.

A new method for the simultaneous solubilization and separation of different cation dependent adenosinetriphosphatases after non-denaturing polyacrylamide gel electrophoresis is described. Using a gradient system, 3 distinct divalent cation dependent adenosinetriphosphatase bands (Mg2+-, Ca2+-, and Mg2+ + Ca2+-dependent) could be separated on the same gel from NP-40 solubilized brain microsomal preparations. This indicates that these 3 adenosinetriphosphatases represent distinct molecular species.

Animals↗
PubMed1983

A microradioiodination method for detection of nanogram amounts of proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A procedure is described for the rapid analysis of nanogram quantities of protein resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Proteins are first radioiodinated by the chloramine T method, acid precipitated after addition of a visible marker protein and collected on a nitrocellulose filter. The region of the filter containing protein is excised and the proteins are then extracted from the filter into electrophoresis sample buffer.

Autoradiography↗
PubMed1986

Routine detection of human rotavirus by latex agglutination: comparison with enzyme-linked immunosorbent assay, electron microscopy and polyacrylamide gel electrophoresis.

A commercially available latex agglutination test, RotaScreen (Mercia Diagnostics Ltd., West Byfleet, Surrey, U.K.) was evaluated for the detection of human rotaviruses in stool specimens. The results obtained were compared with those from 3 other routine assay systems used in this laboratory: enzyme-linked immunosorbent assay (ELISA), electron microscopy (EM) and polyacrylamide gel electrophoresis (PAGE) of viral ribonucleic acid. 400 stool samples were examined by the 4 assay systems under routine conditions. RotaScreen latex agglutination was found to be more sensitive than EM and PAGE, and highly specific for rotavirus antigens.

Adult↗
PubMed1994

Polyacrylamide gel electrophoresis of viral genomic RNA as a diagnostic method for infectious pancreatic necrosis virus detection.

A rapid and simple technique for the diagnosis of IPNV from cell culture and infected fish tissues has been developed. It is based on the observation of IPNV bisegmented double-stranded RNA genome in silver stained polyacrylamide gels after electrophoresis. The method is highly specific and can detect as little as 1 ng of viral RNA, which corresponds to 1 x 10(5) pfu, making it possible to visualize the viral genome as soon as the initial cythopatic effect appears. Furthermore, the RNA viral genome could be detected directly from fish tissues when fish showed clear clinical signs of the disease. The method has the advantage of detecting any strain of IPNV. An acrylamide concentration of 6% and bisacrylamide concentration of 0.13% give a rapid and definitive result in less than 6 h.

Animals↗
PubMed1982

Interaction between the alpha 1 chain of rat tail collagen and sodium dodecyl sulfate with reference to its behaviour in SDS-polyacrylamide gel electrophoresis.

The interaction between the alpha 1 chain of rat tail collagen and sodium (SDS) was studied to provide knowledge necessary to understand the behavior of the complex between them in a molecular sieving technique such as SDS-polyacrylamide gel electrophoresis. Measured properties include binding isotherm, CD spectrum, viscosity and behavior in free boundary electrophoresis, gel electrophoresis and high-pressure silica gel chromatography. The complex differed in most respects from the complexes between SDS and polypeptides derived from water-soluble globular proteins, reflecting its peculiar amino acid composition. THe hydrodynamically effective volume of the SDS-alpha 1 chain complex was the most significantly different among the properties studied. The complex of the alpha 1 chain consisted of 1052 amino acid residues and was found to have the same effective volume as that of a standard polypeptide consisting of about 800 residues. The abnormal behaviour of the SDS-alpha 1 chain complex was interpreted on the basis of these results.

Animals↗
PubMed1982

Subcellular fractions of the adrenal medulla. Comparison by two-dimensional polyacrylamide gel electrophoresis.

Subfractions of adrenal medullary homogenates were analyzed in two-dimensional polyacrylamide gels to assess the extent of protein homology. Chromaffin granule proteins were highly acidic, with the exception of the soluble form of the enzyme dopamine beta-hydroxylase (EC 1.14.17.1). The purified granule membrane proteins were more heterogeneous, but still predominantly acidic. The soluble and membrane forms of dopamine beta-hydroxylase behaved identically in this gel system. Lactoperoxidase-catalyzed iodination of intact granules revealed that most, but not all, granule membrane proteins are accessible at the cytoplasmic face. Prominent proteins of the purified adrenal medullary mitochondria showed little if any homology with purified granule membranes. The crude microsome fraction showed significant homology with purified granule membranes despite low levels of cross-contamination between the two fractions in marker enzyme analysis. Among proteins that could be identified, dopamine beta-hydroxylase was at a low level in the microsomes, while the granule membrane protein cytochrome b-561 appeared to be in both fractions. The pattern obtained from primary cultures of adrenal chromaffin cells was very complex, but prominent proteins from the subcellular fractions were seen without difficulty. Actin and tubulin were very prominent in the whole cell pattern. Radioiodination of the whole cells resulted in a number of spots being labelled, although the majority of the label appeared to be in only two proteins of molecular weight 70000 and isoelectric point 5.7.

Adrenal Medulla↗
PubMed1982

Preparative electrophoresis of histones in solubilizable polyacrylamide gels.

Histones from the mouse testis have been fractionated on 17% acrylamide gels containing 0.19% Bis-acrylylcystamine (BAC), 25 M urea and 0.4% Lubrol-WX at pH 2.7. Polyacrylamide gels which contain BAC, a cross-linking agent with disulfide bonds, can be solubilized in the presence of 2-mercaptoethanol, at pH 8.3. Polymerization is carried out at 40 degrees C and in the presence of 6.5--7.5% tetramethylethylenediamine (TEMED) at pH 8.3. Gels containing high acrylamide concentration (greater than 15%) are not soluble if polymerized at lower pH or TEMED concentrations. Following electrophoresis, the gels are stained with Coomassie brilliant blue R for the visualization of protein bands. The stained protein bands are excised and adjusted to pH 8.3 prior to their solubilization. The histones are recovered by ion-exchange chromatography and are free of soluble gel components and dye. Two variants of histone H2B (H2B . 1 and H2B . 2) have been isolated at approx. 95% purity using this single purification step.

Amino Acids↗
PubMed1988

Investigation of the physical properties of dog intestinal microvillar membrane proteins by polyacrylamide gel electrophoresis: a comparison between normal dogs and dogs with exocrine pancreatic insufficiency.

Procedures have been validated for the investigation of the physical properties of canine microvillar membrane proteins by SDS-polyacrylamide gel electrophoresis. These have been used to examine mucosal samples from eight control dogs and from five dogs with naturally occurring exocrine pancreatic insufficiency (EPI) in order to evaluate the potential role of the pancreas in the normal turnover of microvillar membrane proteins in the dog. Gel scanning showed that the proportion of total membrane protein in bands corresponding to a molecular mass greater than 200 kDa was up to 20-times higher in dogs with EPI than in control dogs. In particular, a band of apparent molecular mass 218 kDa represented between 8 and 28% of membrane protein in all affected dogs, compared with only 0.5 to 1.8% in controls, and is most likely to contain single chains of both pro-maltase-glucoamylase and pro-sucrase-isomaltase. Incubation of microvillar membranes in vitro with either trypsin or canine pancreatic juice resulted in degradation of this high molecular mass band and a corresponding increase in the amount of protein in three bands representing molecular masses of 150, 133 and 106 kDa. In samples from control dogs aminopeptidase N was identified in the 133 kDa band by Western blotting and incubation with monospecific antiserum. These findings suggest that pancreatic enzymes play a major role in the normal post-translational processing of intestinal microvillar membrane proteins in the dog.

Animals↗
PubMedDate not supplied

A silver impregnation method for labeling both Alzheimer paired helical filaments and their polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The Gallyas silver impregnation which is specific to neurofibrillary changes of paired helical filaments (PHF) and 15 nm straight filaments, was adapted to stain polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both PHF and tau polypeptides were readily and consistently stained by the Gallyas stain. This technique stained PHF greater than tau greater than high-molecular-weight microtubule-associated polypeptides (MAPS). Tubulin was stained only weakly. Neurofilament triplet, ubiquitin, bovine serum albumin and histones were unstained. The staining of PHF and tau polypeptides by Gallyas silver stain is consistent with the presence of tau in PHF.

Alzheimer Disease↗
PubMed1986

Specific activity staining for prostaglandin metabolizing enzymes on polyacrylamide gel.

A simple activity staining technique on polyacrylamide gel for 15-hydroxyprostaglandin metabolizing enzymes was developed in which production or digestion of 15-keto-PGE was stained. The rapidity and specificity of this staining was shown with a preparation of NAD-linked 15-hydroxyprostaglandin dehydrogenase isolated from human term placenta.

Clinical Enzyme Tests↗
PubMed1985

Hormonal regulation of proteins secreted by cultured pig Sertoli cells: characterization by two-dimensional polyacrylamide gel electrophoresis.

Using a primary culture of immature porcine Sertoli cells, we studied the effect of porcine FSH (pFSH), testosterone and retinoic acid on the labelled secreted protein. Cells were cultured in a chemically defined medium for 3 days and, on day 3, they were incubated for different times in another medium containing labelled amino acids either in the presence or absence of pFSH (50 ng/ml or 2 micrograms/ml), or testosterone (10(-6) M), or retinoic acid (10(-7) M), either alone or in several combinations. After 4 and 8 h of incubation, 20 and 30 secreted peptides were detected respectively by a two-dimensional polyacrylamide gel electrophoresis of the radiolabelled secreted proteins. During these 2 periods, the effect of pFSH was negligible. After 25 h, about 84 spots (pI in the range of 5-8) were identified on the autoradiograms. pFSH (2 micrograms/ml) induced an increase of 14 spots, and a decrease of 7, but at 50 ng/ml only 5 spots were increased and one decreased. Retinoic acid alone induced the increase of one peptide, while testosterone alone or in combination with pFSH (50 ng/ml) did not modify the electrophoretic pattern. When pig Sertoli cells were treated with retinoic acid, testosterone and pFSH (50 ng/ml), the effects on secreted proteins were higher than those induced by pFSH (50 ng/ml) alone.

Animals↗
PubMed1978

Lipopolysaccharide interferes with the staining of lipoprotein on polyacrylamide gels.

After electrophoresis of total membrane preparations of Escherichia coli B on sodium dodecyl sulfate polyacrylamide gels, and subsequent staining with Coomassie Brilliant blue, a band corresponding to the Braun lipoprotein fails to appear. This is in contrast to similar preparations of E. coli K-12 which do display the lipoprotein upon staining. Experiments described below indicate that failure to observe this protein in E. coli B is due to interference in the staining reaction by the lipopolysaccharide present in the membrane preparations.

Bacterial Proteins↗
PubMed1982

Identification of protein bands in polyacrylamide gel by protein printing.

Previously described methods for identification of proteins separated in cylindrical polyacrylamide gels have been found to be costly in time and antiserum and difficult to apply to small amounts of protein as are found in cerebrospinal fluid. We describe a method which involves printing of the proteins on the cut surface of the gel onto nitrocellulose paper. The protein bands of the imprint can then be identified using labelled antibodies. We have found this to be economical and quick, and it has permitted sensitive and reliable identification of proteins in unconcentrated cerebrospinal fluid and aqueous humour.

Blood Proteins↗
PubMed1984

Monooxygenase activity of rat liver microsomes immobilized by entrapment in a crosslinked prepolymerized polyacrylamide hydrazide.

Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.

Acrylic Resins↗
PubMed1988

Isolation of a component from commercial coomassie brilliant blue R-250 that stains rubrophilin and other proteins red on polyacrylamide gels.

Commercially available Coomassie Brilliant Blue R-250 (C.I. 42660) is a popular and useful dye that stains most proteins blue on polyacrylamide gels. Some proteins from brain (rubrophilin), collagens, histones and parotid gland proteins are distinctly red when stained with Coomassie Blue. Commonly used Coomassie Brilliant Blue R-250 preparations may contain more than 30 distinct colored and fluorescent components that can be separated on silica gel chromatographic columns. A specific component has been isolated on silica gel columns that stains rubrophilin and other proline-rich proteins a reddish color. Fast atom bombardment mass spectrometry of the isolated rubrophilin staining principle indicates a molecular weight of 634 as compared to 826 for the major dye in the original Coomassie Brilliant Blue R-250. Infrared spectrometry is consistent with a difference between the rubrophilin staining principle and Coomassie Brilliant Blue R-250 of a toluene sulfonic acid residue.

Acrylic Resins↗
PubMed1978

Cytoplasmic RNA from hen reticulocytes, mouse sarcoma 180 ascites cells, rat liver and barley embryos. Their preparation and purification by a standard procedure and characterization by polyacrylamide gel electrophoresis.

1. This paper describes a standard procedure for the preparation and purification of RNA from the post-mitochondrial supernatants of a number of eukaryotes. 2. Cytoplasmic RNA was fractionated by NaCl precipitation. The 28S (26S), 18S and 5.8S rRNA, and 9S RNA, in the NaCl insoluble fraction were separated by a two-step sucrose gradient fractionation procedure. Poly(A)-containing mRNA in hen 9S RNA was purified by affinity chromatography. The 5S rRNA and tRNA in the NaCl-soluble fraction were fractionated by gel filtration. 3. Polyacrylamide gel electrophoresis showed that the above RNA species were remarkably stable and homogeneous. Differences were found in the 26-28S rRNA, 5.8S rRNA, and 9S RNA of different eukaryotes, but other cytoplasmic RNA species were identical.

Animals↗
PubMed1984

Detection and characterization of epidermal proteinases by polyacrylamide gel electrophoresis.

Electrophoresis of cornified cell extracts of 2-day-old rats, using SDS polyacrylamide gels copolymerized with alpha-casein or gelatin with or without plasminogen, was performed. Both Tris-HCl buffer soluble protein and KSCN solubilized protein contained a number of hydrolases for alpha-casein and/or gelatin, but PA (mol. wts 57 and 50K) was found only in the KSCN extract. The pH dependency, substrate specificity and mol. wt of plasminogen-independent proteinases were comparatively determined and DFP inhibition tested. This simple technique helped to identify the presence of several proteinases and to characterize them in partially purified epidermal extracts.

Animals↗